Cultivation of mammalian cells is one of many processes in the life and health sciences. Vessels for mammalian cell culture and analysis involving anchorage-dependent cells are often made of glass or a polymer, such as, for example, polystyrene, that frequently requires additional surface treatment to allow the cells to attach to the surface of the vessel. Such treatments may include applying an adlayer on the surface, for example, by adsorption, grafting or plasma polymerization techniques. Alternatively, the surface treatment may be via chemical modification of the vessel surface itself, which can be achieved by, for example, atmospheric corona, radio frequency vacuum plasma, DC glow discharge, and microwave plasma treatments.
Current methods of culturing pluripotent stem cells, in particular, embryonic stem (ES) cells require complex culture conditions, such as, for example, culturing the embryonic stem cells on a solid substrate surface with a feeder cell layer, or on a solid substrate surface with an adlayer of extracellular matrix protein. Culture systems that employ these methods often use feeder cells or extracellular matrix proteins obtained from a different species than that of the stem cells being cultivated (xenogeneic material). Media obtained by exposure to feeder cells, that is, media conditioned by cells other than undifferentiated ES cells, may be used to culture the ES cells, and media may be supplemented with animal serum.
For example, Reubinoff et al. (Nature Biotechnol. 18:399-404, 2000) and Thompson et al. (Science 282:1145-1147, 1998) disclose the culture of ES cell lines from human blastocysts using a mouse embryonic fibroblast feeder cell layer.
In another example, Xu et al. (Nature Biotechnology 19: 971-974, 2001) discloses the use of MATRIGEL® and laminin for treating solid substrate surfaces before feeder-cell free cultivation of human ES cells without differentiation. In another example, Vallier et al. (J. Cell Sci. 118:4495-4509, 2005) discloses the use of fetal bovine serum for treating solid substrate surfaces before feeder-cell free cultivation of human ES cells without differentiation.
In another example, WO2005014799 discloses conditioned medium for the maintenance, proliferation and differentiation of mammalian cells. WO2005014799 state: “The culture medium produced in accordance with the present invention is conditioned by the cell secretion activity of murine cells, in particular, those differentiated and immortalized transgenic hepatocytes, named MMH (Met Murine Hepatocyte).”
In another example, Wanatabe et al. (Nature Biotechnol. 35:681-686, 2007) state “a ROCK inhibitor permits survival of dissociated human embryonic stem cells”, and demonstrate reduced dissociation-induced apoptosis, increases cloning efficiency (from approximately 1% to approximately 27%) and facilitation of subcloning after gene transfer, using mouse embryonic fibroblasts as feeder cells, collagen and MATRIGEL® as extracellular matrix protein, and Y-27632 or Fasudil for inhibition of ROCK. Furthermore, dissociated human ES cells treated with Y-27632 were protected from apoptosis in serum-free suspension culture.
In another example, Peerani et al. (EMBO Journal 26:4744-4755, 2007) state “Complexity in the spatial organization of human embryonic stem cell (hESC) cultures creates heterogeneous microenvironments (niches) that influence hESC fate. This study demonstrates that the rate and trajectory of hESC differentiation can be controlled by engineering hESC niche properties. Niche size and composition regulate the balance between differentiation-inducing and inhibiting factors. Mechanistically, a niche size-dependent spatial gradient of Smad1 signaling is generated as a result of antagonistic interactions between hESCs and hESC-derived extra-embryonic endoderm (ExE). These interactions are mediated by the localized secretion of bone morphogenetic protein-2 (BMP2) by ExE and its antagonist, growth differentiation factor-3 (GDF3) by hESCs. Micropatterning of hESCs treated with small interfering (si) RNA against GDF3, BMP2 and Smad1, as well treatments with a Rho-associated kinase (ROCK) inhibitor demonstrate that independent control of Smad1 activation can rescue the colony size-dependent differentiation of hESCs. Our results illustrate, for the first time, a role for Smad1 in the integration of spatial information and in the niche-size dependent control of hESC self-renewal and differentiation.”
In another example, Koyanagi, M et al (J Neurosci Res. 2008 Feb. 1; 86(2): 270-80) state “Rho-GTPase has been implicated in the apoptosis of many cell types, including neurons, but the mechanism by which it acts is not fully understood. Here, we investigate the roles of Rho and ROCK in apoptosis during transplantation of embryonic stem cell-derived neural precursor cells. We find that dissociation of neural precursors activates Rho and induces apoptosis. Treatment with the Rho inhibitor C3 exoenzyme and/or the ROCK inhibitor Y-27632 decreases the amount of dissociation-induced apoptosis (anoikis) by 20-30%. Membrane blebbing, which is an early morphological sign of apoptosis; cleavage of caspase-3; and release of cytochrome c from the mitochondria are also reduced by ROCK inhibition. These results suggest that dissociation of neural precursor cells elicits an intrinsic pathway of cell death that is at least partially mediated through the Rho/ROCK pathway. Moreover, in an animal transplantation model, inhibition of Rho and/or ROCK suppresses acute apoptosis of grafted cells. After transplantation, tumor necrosis factor-alpha and pro-nerve growth factor are strongly expressed around the graft. ROCK inhibition also suppresses apoptosis enhanced by these inflammatory cytokines. Taken together, these results indicate that inhibition of Rho/ROCK signaling may improve survival of grafted cells in cell replacement therapy.”
The use of xenogeneic material may be unsuitable for certain applications utilizing pluripotent stem cells. Alternative materials may be used. For example, Stojkovic et al. (Stem Cells 23:895-902, 2005) discloses the use of human serum for treating solid substrate surfaces before feeder-cell free cultivation of human ES cells without differentiation.
An alternative culture system employs serum-free medium supplemented with growth factors capable of promoting the proliferation of embryonic stem cells.
For example, Cheon et al. (BioReprod DOI:10.1095/biolreprod.105.046870; 19 Oct. 2005) disclose a feeder-cell free, serum-free culture system in which ES cells are maintained in unconditioned serum replacement medium supplemented with different growth factors capable of triggering ES cell self-renewal.
In another example, Levenstein et al. (Stem Cells 24:568-574, 2006) disclose methods for the long-term culture of human ES cells in the absence of fibroblasts or conditioned medium, using media supplemented with basic fibroblast growth factor (FGF).
In another example, US20050148070 discloses a method of culturing human ES cells in defined media without serum and without fibroblast feeder cells, the method comprising: culturing the stem cells in a culture medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium essentially free of mammalian fetal serum and containing at least about 100 ng/ml of a FGF capable of activating a FGF signaling receptor, wherein the growth factor is supplied from a source other than just a fibroblast feeder layer, the medium supported the proliferation of stem cells in an undifferentiated state without feeder cells or conditioned medium.
In another example, US20050233446 discloses a defined media useful in culturing stem cells, including undifferentiated primate primordial stem cells. In solution, the media is substantially isotonic as compared to the stem cells being cultured. In a given culture, the particular medium comprises a base medium and an amount of each of basic FGF, insulin, and ascorbic acid necessary to support substantially undifferentiated growth of the primordial stem cells.
In another example, U.S. Pat. No. 6,800,480 states: “In one embodiment, a cell culture medium for growing primate-derived primordial stem cells in a substantially undifferentiated state is provided which includes a low osmotic pressure, low endotoxin basic medium that is effective to support the growth of primate-derived primordial stem cells. The basic medium is combined with a nutrient serum effective to support the growth of primate-derived primordial stem cells and a substrate selected from the group consisting of feeder cells and an extracellular matrix component derived from feeder cells. The medium further includes nonessential amino acids, an anti-oxidant, and a first growth factor selected from the group consisting of nucleosides and a pyruvate salt.”
In another example, US20050244962 states: “In one aspect the invention provides a method of culturing primate embryonic stem cells. One cultures the stem cells in a culture essentially free of mammalian fetal serum (preferably also essentially free of any animal serum) and in the presence of fibroblast growth factor that is supplied from a source other than just a fibroblast feeder layer. In a preferred form, the fibroblast feeder layer, previously required to sustain a stem cell culture, is rendered unnecessary by the addition of sufficient fibroblast growth factor.”
In another example, WO2005065354 discloses a defined, isotonic culture medium that is essentially feeder-free and serum-free, comprising: a. a basal medium; b. an amount of basic fibroblast growth factor sufficient to support growth of substantially undifferentiated mammalian stem cells; c. an amount of insulin sufficient to support growth of substantially undifferentiated mammalian stem cells; and d. an amount of ascorbic acid sufficient to support growth of substantially undifferentiated mammalian stem cells.
In another example, WO2005086845 discloses a method for maintenance of an undifferentiated stem cell, said method comprising exposing a stem cell to a member of the transforming growth factor-beta (TGFβ) family of proteins, a member of the fibroblast growth factor (FGF) family of proteins, or nicotinamide (NIC) in an amount sufficient to maintain the cell in an undifferentiated state for a sufficient amount of time to achieve a desired result.
Pluripotent stem cells provide a potential resource for research and drug screening. At present, large-scale culturing of human ES cell lines is problematic and provides substantial challenges. A possible solution to these challenges is to passage and culture the human ES cells as single cells. Single cells are more amenable to standard tissue culture techniques, such as, for example, counting, transfection, and the like.
For example, Nicolas et al. provide a method for producing and expanding human ES cell lines from single cells that have been isolated by fluorescence-activated cell sorting following genetic modification by lentivirus vectors (Stem Cells Dev. 16:109-118, 2007).
In another example, US patent application US2005158852 discloses a method “for improving growth and survival of single human embryonic stem cells. The method includes the step of obtaining a single undifferentiated hES cell; mixing the single undifferentiated cell with an extracellular matrix to encompass the cell; and inoculating the mixture onto feeder cells with a nutrient medium in a growth environment”.
In another example, Sidhu et al. (Stem Cells Dev. 15:61-69, 2006) describe the first report of three human ES cell clones, hES 3.1, 3.2 and 3.3, derived from the parent line hES3 by sorting of single-cell preparations by flow cytometry.
However, passage and culture of human ES cells as single cells leads to genetic abnormalities and the loss of pluripotency. Culture conditions are important in the maintenance of pluripotency and genetic stability. Generally, passage of human ES cell lines is conducted manually or with enzymatic agents such as collagenase, liberase or dispase.
For example, Draper et al. note the presence of “karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem cell lines on five independent occasions.” (Nature Biotechnol. 22:53-54, 2004).
In another example, Buzzard et al. state, “we have only ever detected one karyotype change event . . . the culture methods used may have had some bearing on our results, given that our methods are distinctly different from those used by most other groups. Typically we passage human ES cells after 7 days by first dissecting the colony with the edge of a broken pipette . . . . No enzymatic or chemical methods of cell dissociation are incorporated into this method. We speculate that this may explain the relative cytogenetic resilience of hES (human ES) cells in our hands.” (Nature Biotechnol. 22:381-382, 2004).
In another example, Mitalipova et al. state: “bulk passage methods . . . can perpetuate aneuploid cell populations after extended passage in culture, but may be used for shorter periods (up to at least 15 passages) without compromising the karyotypes . . . it may be possible to maintain a normal karyotype in hES cells under long-term manual propagation conditions followed by limited bulk passaging in experiments requiring greater quantities of hES cells than manual passage methods, alone, can provide”. (Nature Biotechnol. 23:19-20, 2005).
In another example, Heng et al. state “the results demonstrated that the second protocol (trypsinization with gentle pipetting) is much less detrimental to cellular viability than is the first protocol (collagenase treatment with scratching). This in turn translated to higher freeze-thaw survival rates.” (Biotechnology and Applied Biochemistry 47:33-37, 2007).
In another example, Hasegawa et al. state, “we have established hESC sublines tolerant of complete dissociation. These cells exhibit high replating efficiency and also high cloning efficiency and they maintain their ability to differentiate into the three germ layers.” (Stem Cells 24:2649-2660, 2006).
In another example, U.S. Patent application 61/030,544 provides methods and compositions for cell attachment to, cultivation on and detachment from a solid substrate surface containing from at least about 0.9% nitrogen to about at least 11% nitrogen and from at least about 12% oxygen to at least about 30% oxygen, and lacking an adlayer and feeder cells. In one embodiment of the present invention, the cells are treated with a compound capable of inhibiting Rho kinase activity.
There is a significant need for methods and compositions for the culture of cells, including pluripotent stem cells in the absence of feeder cells and an adlayer, while maintaining the pluripotency of the cells. The present invention provides methods for the growth, expansion and differentiation of pluripotent stem cells on planar substrates lacking an adlayer and a feeder cell layer, wherein the cells do not require treatment with a compound capable of inhibiting Rho kinase activity in order to bind to the planar substrate.